Vitamin E reduces high levels of oxysterols and normalizes gene expression in cells from SLOS patients and lowers oxysterol levels in a mouse model of SLOS.

Smith-Lemli-Opitz syndrome (SLOS) is a congenital disorder resulting from mutations in the gene for the enzyme 7-dehydrocholesterol (7-DHC) reductase. People carrying these mutations exhibit multiple congenital malformations, photosensitivity, impaired cognitive functions, and behaviors typical of autism spectrum disorder with varying levels of severity. 7-DHC reductase catalyzes the final step in cholesterol biosynthesis, so mutations resulting in a reduced activity of this enzyme lead to a cholesterol deficiency and a buildup of 7-DHC (Figure 1). Cholesterol is a major lipid component of cellular membranes and serves as the biosynthetic precursor of steroid hormones and bile acids. Thus, the cholesterol deficiency likely contributes to the pathology of SLOS. However, VICB member Ned Porter and his colleagues, Libin Xu (Department of Chemistry), Zeljka Korade and Karoly Mirnics (Department of Psychiatry), and Fiona Harrison (Division of Diabetes, Endocrinology, and Metabolism) propose that excessive levels of 7-DHC also likely play a part. Their hypothesis is based on the high reactivity of 7-DHC to lipid peroxidation. Now, this team of investigators report that antioxidant supplementation helps to reverse some of the molecular defects observed in SLOS, paving the way for a potential therapeutic strategy [Z. Korade, L. Xu, et al. (2013) Biol. Psychiatry, published online July 26, doi:10.1016/j.biopsych.2013.06.013].

Figure 1. Structure of cholesterol and 7-dehydrocholesterol (7-DHC). The conjugated double bond system of 7-DHC confers unusual reactivity to the hydrogen atoms at carbons-9 and 14. Abstraction of either of these hydrogen atoms yields a highly delocalized carbon radical that readily undergoes reaction with oxygen, leading to the formation of multiple products.

7-DHC is the most reactive lipid molecule toward free radical lipid peroxidation that has been tested to date. Due to the conjugated double bonds at positions 5 and 7, the hydrogen atoms at carbons-9 and -14 are highly susceptible to abstraction. Abstraction of either hydrogen yields one of two possible carbon-centered free radicals that is stabilized by delocalization over five atoms (Figure 1). Attack of oxygen on these radicals results in the formation of multiple oxysterol products, including DHCEO, THCEO, and 7-kDHC (full names and structures in Figure 2). Prior studies have revealed increased levels of oxysterols in the cells and tissues of SLOS animal models and demonstrated that these molecules can be toxic. For example, DHCEO alters gene expression, promotes cellular differentiation, and facilitates arborization of mouse cortical neurons. These observations form the foundation for the hypothesis that reducing oxysterol formation by antioxidant treatment should be beneficial to SLOS patients.

Figure 2. Structures of three major 7-DHC-derived oxysterols that have been observed in vivo.

To test their hypothesis, the investigators first treated cultured fibroblasts from SLOS patients with an antioxidant mixture containing vitamins A, C, D, and E, along with coenzyme Q10. The treatment resulted in a concentration-dependent decrease in DHCEO levels in the cells. The antioxidant mixture also increased the expression of four out of six genes involved in lipid metabolism that are suppressed in SLOS cells, as indicated by higher levels of the complementary mRNA in antioxidant-treated cells as compared to controls. For one of the genes, higher levels of the encoded protein were also confirmed.

Further studies indicated that the single active ingredient in the antioxidant mixture was vitamin E. In fact, vitamin E alone completely mimicked the effects of the antioxidant mixture, with DHCEO reduction occurring over a concentration range of 10 nM to 1 μM, and gene expression changes observed at a concentration of 500 nM.

These promising results led the investigators to explore the potential of vitamin E to ameliorate SLOS-associated biochemical abnormalities in vivo. For this purpose, they used Dhcr7 knockout mice, which have been genetically engineered to completely lack the gene for 7-DHC reductase. These mice die soon after birth, so the investigators tested the effects of vitamin E supplementation in utero. Heterozygous male and female Dhcr7(+/-) mice were bred, and the pregnant females were then fed a control diet (no vitamin E), a diet containing standard levels of vitamin E (VEC), or a diet enriched with high levels of vitamin E (VER). Soon after the pups were born, the investigators genotyped the litter. Analysis of the livers and brains of the pups confirmed increased vitamin E levels as a result of both the VEC and VER diets, although the increase was statistically significant only in the case of VER-fed mothers. Of greater interest was the finding that both diets resulted in significant decreases in the levels of DHCEO and THCEO in the brains and THCEO and 7-kDHC in the livers of Dhcr7(-/-) pups.

Together, the results suggest that antioxidant therapy may be of benefit to SLOS patients, although considerable further work is needed. The investigators hope to soon evaluate the effects of antioxidant therapy in an SLOS model that is less severe, so the affected animals survive to adulthood. They also plan to pair antioxidant therapy with cholesterol supplementation to address both of the primary biochemical abnormalities in SLOS. Finally, because vitamin E supplementation can be associated with unwanted side effects, such as fatigue, weakness, headaches, nausea, diarrhea, and bleeding, the team is planning to investigate the effectiveness of novel, nontoxic antioxidants. As antioxidant discovery has been a long-standing interest in the Porter laboratory, work to achieve this goal begins on a firm foundation.